CN102174474B - Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil - Google Patents
Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil Download PDFInfo
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- CN102174474B CN102174474B CN 201110004723 CN201110004723A CN102174474B CN 102174474 B CN102174474 B CN 102174474B CN 201110004723 CN201110004723 CN 201110004723 CN 201110004723 A CN201110004723 A CN 201110004723A CN 102174474 B CN102174474 B CN 102174474B
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- BNRNXUUZRGQAQC-UHFFFAOYSA-N sildenafil Chemical compound CCCC1=NN(C)C(C(N2)=O)=C1N=C2C(C(=CC=1)OCC)=CC=1S(=O)(=O)N1CCN(C)CC1 BNRNXUUZRGQAQC-UHFFFAOYSA-N 0.000 title abstract description 26
- 229960003310 sildenafil Drugs 0.000 title abstract description 11
- 238000004587 chromatography analysis Methods 0.000 title abstract description 6
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Abstract
The invention belongs to the field of medicament inspection and discloses a sildenafil monoclonal antibody and a colloidal gold chromatography test strip used for detecting sildenafil. The sildenafil monoclonal antibody is generated by a hybridoma cell line with the collection number of CCTCC No. C2010123. The sildenafil monoclonal antibody has highly uniform physical and chemical properties, single bioactivity and high specificity of combining the antigen sildenafil, is convenient to perform artificial treatment and quality control, and can be used for preparing the colloidal gold chromatography test strip for detecting the sildenafil. In the colloidal gold chromatography test strip for detecting sildenafil, the gold-labelled antibody coated on a gold-labelled combination pad is the colloidal gold marker of the sildenafil monoclonal antibody. By using the colloidal gold chromatography test strip, no any reagent and instrument are required, and site operation can be performed; and after a sample is mixed with an extraction solution, the supernatant is taken for detection, the detection result can be obtained within 5-10 minutes, and the method is simple, convenient and rapid and has high timeliness.
Description
Technical field
The invention belongs to the drug inspection field, relate to the Virga monoclonal antibody and for detection of the colloid gold chromatographic test paper strip of Virga.
Background technology
Virga (Sildenafil), translate again 'Xiduofeng ', it is a kind for the treatment of male erectile dysfunction medicine that accident is invented out when researching and developing the Cardiovarscular medicine, one is widely known by the people with its commercial name Viagra (China's Mainland login name viagra, Taiwan and Hong Kong login name Vigral) that uses.But with respect to the trade(brand)name Virga the popular name " vigour " of China use more extensive, affect also larger.At present, the illegal Virga that adds, is taken the Chinese patent medicine or the healthcare products that contain Virga and can be caused severe side effect as the patient to reach clinical curative effect in a lot of Chinese patent medicines or the healthcare products in long-term unwitting situation at home.At present, detect in Chinese patent medicine or the healthcare products whether contain the vigour composition, employing be traditional method of inspection, the sample that extracts must be taken back the laboratory and analyze, or use large-scale instruction carriage, so all very inconvenient, and long reaction time, experiment is expensive larger.
Virga belongs to molecular weight less than 1000 micromolecular compound, itself does not possess reactivity and immunogenicity, in order to realize the immunodetection of Virga, not only need the synthetic Virga antigen that possesses high-titer, but also need screening high specificity, Virga monoclonal antibody that susceptibility is high.But the relevant report that does not still have relevant Virga monoclonal antibody at present.
Summary of the invention
The objective of the invention is the above-mentioned deficiency for prior art, the hybridoma cell line of strain secretion Virga monoclonal antibody is provided.
Another object of the present invention provides Virga monoclonal antibody and the application thereof of this hybridoma secretion.
Of the present invention have a purpose to provide a kind of colloid gold chromatographic test paper strip for detection of Virga.
Purpose of the present invention can be achieved through the following technical solutions:
The hybridoma cell line of one strain secretion Virga monoclonal antibody is preserved in Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCC No.C2010123, and preservation date is on November 30th, 2010.
A kind of Virga monoclonal antibody is the hybridoma cell line generation of CCTCC No.C2010123 by preserving number.
The present invention utilizes artificial antigen N-acetic acid Virga-BSA to be immunogen, through Balb/c small white mouse subcutaneous inoculation, the splenocyte and the myeloma cell that get small white mouse behind the booster immunization do cell fusion, screening and cloning, have set up the hybridoma cell strain CCTCC No.C2010123 of stably excreting Virga monoclonal antibody.Through the evaluation of ELISA, Western Blot, colloidal gold immunochromatographimethod method, the monoclonal anti physical efficiency specific recognition Virga that this hybridoma cell strain is secreted.
The application of Virga monoclonal antibody of the present invention in preparation Virga gold-immunochromatographyreagent reagent for assay.
Described Virga detection reagent is Virga detection colloid gold chromatographic test paper strip.
A kind of colloid gold chromatographic test paper strip for detection of Virga, the sample pad that comprises liner plate and be connected successively on liner plate, gold-marking binding pad, coated film and absorbent pad form, gold-marking binding pad is the trevira felt of absorption Virga gold labeling antibody, at coated film the coated orthoscopic detection line T line of N-acetic acid Virga-BSA antigenic solution is arranged, with the orthoscopic control line C line that is coated with sheep anti-mouse igg solution, article two, line is vertically arranged in parallel, and described Virga gold labeling antibody is the colloid gold label thing of Virga monoclonal antibody of the present invention.
Described Virga antigen is the Virga artificial antigen.
Described Virga artificial antigen is:
(1), wherein Pro is carrier proteins, preferred bovine serum albumin, chicken ovalbumin or people's hemocyanin, further preferred bovine serum albumin or chicken ovalbumin, particularly preferably bovine serum albumin, i.e. N-acetic acid Virga-BSA.
The preparation method of the colloid gold chromatographic test paper strip for detection of Virga of the present invention comprises following steps:
(1) the Virga artificial antigen shown in the preparation formula (1);
(2) preparation Virga monoclonal antibody;
(3) preparation contains the mixing solutions of the colloid gold label thing of the colloid gold label thing of anti-Virga monoclonal antibody and mouse IgG monoclonal antibody;
(4) utilize the mixing solutions of the colloid gold label thing of the described colloid gold label thing that contains anti-Virga monoclonal antibody of step (3) and mouse IgG monoclonal antibody to prepare the gold-marking binding pad of Virga monoclonal antibody;
(5) be coated with orthoscopic detection line T line at coated film with described Virga artificial antigen solution, with the coated orthoscopic control line C line of sheep anti-mouse igg solution, two lines are vertically arranged in parallel;
(6) assembling test strip.
Wherein, the preparation method of the Virga artificial antigen shown in the formula (1) sees Chinese patent 201010248466.5 for details.
The mixing solutions that step (3) is described to contain the colloid gold label thing of the colloid gold label thing of anti-Virga monoclonal antibody and mouse IgG monoclonal antibody prepares by the following method: the colloidal gold solution of the hydrochloro-auric acid reduction being made 20nm-40nm with the trisodium citrate reductive agent; Use 0.1mol/LK
2CO
3Regulate described colloidal gold solution to optimal pH 8.5-9.0, after the Virga monoclonal antibody pure water dialysis of the present invention with purifying, add in the colloidal gold solution by 3 μ g/ml amount, room temperature induction stirring 15-30min, then the amount by 5 μ g/ml adds mouse IgG monoclonal antibody, room temperature induction stirring 15-30min, dropwise add 1% network protein solution, making final concentration is 0.1%, continue to stir 5min, 4 ℃ of centrifugal 10min of lower 15000rpm, supernatant discarded, throw out is returned to the original volume mixing again with resuspended liquid, recentrifuge once is suspended to 1/20,4 ℃ of original volume with resuspended solution with throw out and saves backup, namely obtain the mixing solutions of the colloid gold label thing of the described colloid gold label thing that contains anti-Virga monoclonal antibody and mouse IgG monoclonal antibody, wherein said resuspended liquid is the 0.02Tris-HCL damping fluid that contains 5% marine alga;
The method of the gold-marking binding pad of the described preparation Virga of step (4) monoclonal antibody is: with 1: 20-1: the mixing solutions spraying of the colloid gold label thing that contains anti-Virga monoclonal antibody of 10 dilutions and the colloid gold label thing of mouse IgG monoclonal antibody is adsorbed in the trevira felt 27 ℃ of heat preservation and drynesses;
The method of the described assembling test strip of step (6) is: stick to successively sample pad, gold mark combination mat, coated film and absorbent pad on the liner plate, sample pad places an end of liner plate, absorbent pad places the other end of liner plate, coated film places the centre of liner plate, gold-marking binding pad places between sample pad and the coated film, namely obtain detecting the large plate of test pater for the Virga colloidal gold chromatographic of immunodetection, large plate is cut into the test strip of 2.5mm-7.0mm width specifications by customer requirement, hermetically drying is preserved.
The present invention compared with prior art has the following advantages:
1, Virga monoclonal antibody physicochemical property height homogeneous of the present invention, biological activity high specificity single, that be combined with antigen, be convenient to artificial the processing and quality control, and the source is easily.
2, colloidal gold chromatographic test strip of the present invention is prepared from as the basis take the monoclonal antibody of colloid gold label high specific high-affinity electrostatic adhesion, has high specificity, the advantage that susceptibility is high can specificly detect and whether contain Virga in the sample and have the compound of identical parent nucleus with Virga.
3, use colloidal gold chromatographic test strip of the present invention, need not any other reagent and instrument, but execute-in-place after sample mixed with extracting solution, get supernatant liquor and detect, can judge detected result at 5min, easy and simple to handle, ageing strong.
4, colloidal gold chromatographic test strip result of the present invention shows image, directly perceived, accurate.Test strip is all to show that red line T line and C line are as the detected result positive and negative marker, namely when coated film shows a red line C line, be illustrated in and contain checking matter in the test sample, when showing two red line T lines and C line, be illustrated in to be subtracted and do not contain checking matter in the sample.The result judges image, directly perceived, accurate, simple and clear, is not prone to the artificial erroneous judgement such as false positive and false negative.
5, the preparation method of test strip of the present invention adopts spraying coating process, Simple fast, and spraying is even, fast drying.
Biomaterial preservation information
Hybridoma cell strain 2H5B12C3 of the present invention on November 30th, 2010, is preserved in Chinese Typical Representative culture collection center, is called for short CCTCC, and the address is Wuhan City Wuhan University, and deposit number is CCTCC NO:C2010123.
Description of drawings:
Fig. 1 is the cross-sectional view of colloid gold chromatographic test paper strip of the present invention;
1-sample pad, 2-gold-marking binding pad, 3-coated film, 4-T line, 5-C line, 6-absorbent pad, 7-liner plate.
Fig. 2 is the plan structure schematic diagram of colloid gold chromatographic test paper strip of the present invention;
1-sample pad, 2-gold-marking binding pad, 3-coated film, 4-T line, 5-C line, 6-absorbent pad.
The result of five concentration gradients of Fig. 3 Virga 3.0mm test card inspection Virga.
Fig. 4: the result of five concentration gradients of Virga 3.0mm test card inspection Vardenafil.
Fig. 5: the result of five concentration gradients of Virga 3.0mm test card inspection Acctildenafil.
Fig. 6: the result of five concentration gradients of Virga 3.0mm test card inspection Tadalafei.
Fig. 7, N-acetic acid Virga nuclear magnetic resonance map;
Fig. 8, N-acetic acid Virga mass spectrum;
Fig. 9, N-acetic acid Virga HPLC collection of illustrative plates;
Figure 10, Western Blot immunoblotting scintigram, M is Maker, the 1st, N-acetic acid Virga-BSA, the 2nd, BSA is as negative control.
Embodiment
A synthesizes N-acetic acid Virga (II-i)
(1) alkylation: the dimethyl formamide (DMF) and the 1.708g, 3.6 * 10 that add the 20ml drying in the reaction flask
-3The Virga of mol (IV-i), and logical N
2Protection is at room temperature stirred, and after dimethyl formamide (DMF) dissolves fully, takes by weighing fast 0.173g, 4.32 * 10
-3Mol, concentration are 60% sodium hydride (NaH), and it is added in the described reaction flask, stir 60 minutes, until solution without Bubble formation, is then drawn 1.202g, 7.2 * 10 with the 1ml syringe
-3The ethyl bromoacetate of mol is slowly injected described reaction flask, inject complete after, at room temperature stirred 12 hours, will disperse in the reaction solution impouring 60ml water afterwards, extract with the chloroform of 20ml * 3, united extraction liquid is with anhydrous Na
2SO
4Carry out drying, filter afterwards, obtain solid through behind the concentrating under reduced pressure again, add the described solid of 6ml anhydrous diethyl ether making beating washing, filter afterwards, last vacuum-drying obtains the new solid of 1.722g, yield 85.31%, this solid are the alkylide (formula (V-i)) of Virga;
(2) hydrolysis: with the 1.682g, 3.0 * 10 of 15ml dehydrated alcohol and above-mentioned steps gained
-3The Virga alkylide of mol (formula (V-i)) adds in the new reaction flask, carries out dispersed with stirring, adds the NaOH solution of 10ml, 1mol/L again, heating reflux reaction 2 hours, after stopped heating slightly cools off, reaction solution is changed in the single port bottle, concentrating under reduced pressure gets mashed prod, add again 20ml water, be adjusted to the liquid of pH value as 8 take 3N hydrochloric acid, extract united extraction liquid with the chloroform of 15ml * 3, again with the saturated common salt washing once, with anhydrous Na
2SO
4Drying is filtered, and concentrating under reduced pressure gets white solid, uses recrystallizing methanol, gets 1.222g white powdery solid, and yield 76.47%, this solid are N-acetic acid Virga (formula (II-i));
Hapten compound N-acetic acid Virga molecular weight is 532.6, and fusing point is 136 ℃, and nucleus magnetic resonance verifies that its structure is formula (II-i), sees Fig. 7 for details; MS:533.22 (M+H
+) see Fig. 8 for details; It is 99.78% that HPLC detects purity, sees Fig. 9 for details.
Concrete route is as follows:
B, generation artificial antigen:
(1) with the N-acetic acid Virga (formula (II-i)) of gained in the above-mentioned steps, N-hydroxy-succinamide (NHS), dimethyl formamide (DMF), water-soluble carbodiimide (EDC) balance to room temperature;
(2) get N-acetic acid Virga (formula (II-i)), EDC and NHS, three's mol ratio is: N-acetic acid Virga: EDC: NHS=1: 10: 15, it is inserted the in advance round-bottomed flask of wash clean, the an amount of dimethyl formamide dissolving that adds gained in the above-mentioned steps, one is according to the dimethyl formamide that how much adds of above-mentioned mixing liquid, the add-on of dimethyl formamide is little on the reaction impact, one adds the 2-4ml dimethyl formamide, can dissolution sample get final product, influential to the protein carrier in next step reaction too much, for guaranteeing to react completely, under the condition of room temperature lucifuge, stir 24h, the solution that generates at last is designated as reaction solution No. two for containing the reaction solution of formula (III-i) compound.Wherein can not there be water residual in the flask, needs oven dry and be chilled in advance room temperature with front, in order to avoid affect the stability of reactant, product, affect reaction efficiency;
(3) after N-acetic acid Virga, EDC and NHS react completely in dimethyl formamide, existing prestowage body protein solution bovine serum albumin solution (BSA solution), bovine serum albumin and borate buffer are dissolved in borate buffer in another clean container by the amount of 1: 0.15 (100mg is dissolved in the 15ml borate buffer), then change in another clean round-bottomed flask, this solution is designated as reaction solution No. three, wherein borate buffer and carrier proteins reaction solution must be now with the current, and borate buffer is pH9.0;
(4) No. two reaction solutions are splashed in No. three reaction solutions by per 10 seconds speed of 1, until after No. two reaction solution added, wherein carrier proteins BSA and formula (III-i) compound ratio was: 1: 20.Stirred 2 hours under the condition of room temperature lucifuge first, lucifuge stirred 48 hours under 3-5 ℃ of condition afterwards, and reaction generates the artificial antigen that N-acetic acid Virga-BSA (formula (I-i)) is Virga.
(5) etc. after institute responds and finishes, the reaction solution of step (4) gained is placed 20000 molecular retention dialysis tubings, the 0.01M phosphate buffered saline buffer also is housed in the dialysis tubing, the pH value is 7.0-7.5, with the dialysis tubing 48h that under 3-5 ℃ condition, dialyses, changed one time phosphate buffered saline buffer every 3 hours;
(6) sample of gained in the dialysis above-mentioned steps is white cotton-shaped product through lyophilize, is the artificial antigen N-acetic acid Virga-BSA of purified Virga.
Concrete route is as follows:
The foundation of embodiment 2 hybridoma cell lines
Artificial antigen N-acetic acid Virga-BSA adds the Freund's complete adjuvant emulsification of equivalent, minute multiple spot mouse subcutaneous injection 0.2ml (1mg/ml).After 3 weeks, after N-acetic acid Virga-BSA adds the abundant mixing of Freund's incomplete adjuvant of equivalent, minute multiple spot mouse subcutaneous injection 0.2ml (1mg/ml), after 10 days, one of blood is got in docking, surveys antibody titer, selects the high mouse of titre to do test for fusion.Can give without adjuvant antigen N-acetic acid Virga-BSA0.1ml through vein after one month, after 3 days, kill mouse and get spleen and do and merge to use.
Small white mouse myelomatosis system derives from Balb/c small white mouse system.Balb/c is what small white mouse must be with pure lines, male and female all can, to be advisable ages in 8～12 weeks.
Being prepared as follows of splenocyte:
(1) the immune high Balb/c mouse of serum antibody titer that crosses draws neck to put to death small white mouse.
(2) mouse is put in soaking disinfection in 70% alcohol, takes out and be fixed on the plate, under aseptic condition, get spleen.
(3) spleen is put into 1640 liquid that 5ml contains 2.5%FCS, the red blood corpuscle on the flush away spleen gently under the ice bath.
(4) push gently spleen with tweezers, make splenocyte suspension, with kapillary suspension is moved in the small test tube.
(5) upright small test tube 3min sinks the reticular tissue of bulk, and cell suspension is moved in the centrifuge tube.
(6) be full of centrifuge tube with 2.5%FCS-1640, and with the centrifugal 10min of 400g.
(7) precipitation is suspended with the about fresh medium of 10ml again.
(8) repeat (6), (7) step.
(9) calculate cell, expect that with platform it is qualified that blue dyeing uses phase-contrast microscopy, viable count should be higher than 80%.
(1) gets the 3rd day mouse boosting cell suspension after the last immunity, with 10
8Mouse boosting cell and 3 * 10
7Murine myeloma cell is mixed in 50 milliliters of graduated centrifuge tubes, and is centrifugal 7 minutes through 1000 rev/mins;
(2) abandon supernatant liquor, remove as far as possible totally, with finger tapping centrifuge tube bottom, make precipitation mixing such as pasty state, centrifuge tube is put and is carried out water-bath (in the small beaker) in 37 ℃ of water, prepares to merge;
(3) the 50%PEG1.0 milliliter that is incubated in 37 ℃ of water-baths is slowly splashed in the centrifuge tube with dropper, within 60 seconds, drip off, in 37 ℃ of water-baths, shake the limit centrifuge tube while dripping, make cell be kept at the mixing state;
(4) add PEG after, cell suspension is placed in 37 ℃ of water-baths left standstill for 90 seconds, in 3 minutes, add immediately the RPMI-1640 substratum (37 ℃) of 1 milliliter of serum-free, begin and will drop by drop add, because dilution fails PEG.Note not stirring as far as possible cell;
(5) centrifugal 10 minutes through 100 rev/mins again.Abandoning supernatant adds 25 milliliters of HAT nutrient solutions, and mixing has the suspension packing in two 24 well culture plates of scavenger cell 0.5 milliliter in every hole gently;
(6) culture plate is placed on the saturated CO of water vapor
2In the incubator, hatch CO for 37 ℃
2Content is 5%;
(7) changed one time the HAT nutrient solution in per 2 days, whether continuous 2 weeks observe hybridoma and occur.Use the HT substratum after 2 weeks.
Step 3ELISA experiment screening positive hybridoma cell
(1) N-acetic acid Virga-BSA is diluted to 1-20ug/ml with coating buffer.
(2) add in the enzyme plate hole with 50ul/ hole amount, put 4 ℃ and spend the night or 37 ℃ of absorption 2 hours.
(3) discard liquid in the hole, wash 3 times with washings simultaneously, each 3-5 minute, pat dry.
(4) every hole adds that 4 ℃ of 200ul confining liquids spend the night or 37 ℃ of sealings 2 hours.
(5) washings is washed 3 times; This moment, coated plate can-20 ℃ or 4 ℃ saves backup.
(6) every hole adds 50ul Hybridoma Cell Culture supernatant to be checked, sets up simultaneously the positive, negative control and blank; Hatched 1-2 hour for 37 ℃; Washing pats dry.
(7) enzyme-added mark second antibody, every hole 50-100ul was hatched 2 hours for 37 ℃, and washing pats dry.
(8) add substrate solution, every hole adds freshly prepared substrate and uses liquid 50-100ul, 37 ℃ 30 minutes.
(9) with 2mol/L H
2SO
4Termination reaction reads the OD value at the enzyme linked immunological reading apparatus.
(10) result judges: with P/N 〉=2.1, or P 〉=N+3SD is positive.If negative control hole is colourless or near colourless, clearly develop the color in the positive control hole, the result then can directly detect by an unaided eye.
The cloning of step 4 positive hybridoma cell
(1) preparation Peritoneal Cells of Mice is with the method in " cytogamy " joint.
(2) prepare hybridoma suspension to be cloned, with the HT substratum that contains 20% serum be diluted to every milliliter contain 2.5,15 with 3 kinds of different extent of dilution of 50 cells.
(3) add 5 * 10 by every milliliter
4The ratio of cell adds respectively peritoneal macrophage in above-mentioned hybridoma suspension.
(4) every kind of hybridoma packing 96 orifice plate are one, each extent of dilution 32 hole, and every hole amount is 0.2ml, the hybridoma number in every hole is respectively 0.5,3 and 10.
(5) 37 ℃, 5%CO
2Moistening cultivation 9 days macroscopic clone occurs and can detect antibody; Under inverted microscope, observe, mark the hole of only having single clonal growth, get supernatant and do antibody test.
(6) get the cell in the positive hole of antibody test, namely obtain the monoclonal antibody secrete strain, a large amount of amplifications are also frozen, after Long Term Passages is cultivated, with identical method again cloning identify it, thereby obtained the hybridoma cell strain 2H5B12C3 of stably excreting Virga monoclonal antibody.Hybridoma cell strain 2H5B12C3 is preserved in Chinese Typical Representative culture collection center (being called for short CCTCC) on November 30th, 2010, and preserving number is CCTCC NO:C2010123.
(7) (2), (3), (4) also can be reduced to the hybridoma behind the counting is carried out serial dilution exactly in this law, until every milliliter contain 10 cells, by every hole inoculation 0.1ml cell suspension, namely every hole contains 1 cell.
Induce the standby monoclonal antibody of ascites legal system in embodiment 3 bodies
Generally all adopt the method for producing monoclonal antibody in the animal body, in view of most animals are merged with splenocyte with strain by the myeloma cell of Balb/c mouse with hybridoma and get, the therefore certain first-selected Balb/c mouse of animal of use.Present method is about to hybridoma and is inoculated in the mouse peritoneal, the hybridoma of in mouse peritoneal, growing, and produce ascites, thereby can obtain a large amount of ascites monoclonal antibodies and antibody concentration very high.As seen this method is easy and simple to handle, economical, but, often is mixed with the various foreign proteins of mouse in the ascites, could use after therefore will purifying under many circumstances, and pollute in addition the danger of animal virus, so the most handy SPF level mouse.Working method is as follows:
(1) intraperitoneal inoculation whiteruss, every mouse (SPF level) 0.5ml.
The hybridoma that pneumoretroperitoneum inoculation in (2) 8 days is diluted with serum free medium, every mouse 5 * 10
5/ 0.2ml.
(3) every day was observed the mouse ascites production after 5 days in the interval, obviously expanded such as belly, and when touching with hand, skin has tension, and namely available No. 16 syringe needles gather ascites, and one can be adopted 2 times continuously, and every mouse can be adopted 8ml ascites usually;
(4) with ascites centrifugal (2000r/min 5 minutes), remove cellular constituent and other throw out, collect supernatant, measure antibody titer, packing ,-70 ℃ are frozen for subsequent use, or freeze-drying is preserved.
Embodiment 4 monoclonal antibody CHARACTERISTICS IDENTIFICATION
4.1 the titer determination of monoclonal antibody
The titer determination of monoclonal antibody can adopt agglutination reaction, ELISA or radioimmunoassay.Different measuring method titres is different, adopts agglutination reaction, and the ascites titre of monoclonal antibody of the present invention can reach 5 * 10
4And adopting ELISA to check, the ascites titre of monoclonal antibody of the present invention can reach 1.0 * 10
6
4.2 the classification of immunoglobulin (Ig) and subgroup identification
Method: with the antigen N-acetic acid Virga of suitable concentration-BSA coated elisa plate, the 100ul/ hole, 4 ℃ are spent the night.After the washing, add monoclonal antibody sample to be checked, the 100ul/ hole, 37 ℃ 1 hour; If negative, positive control hole.After the washing, add the anti-little muroid of HRP mark and the antibody reagent of subclass Ig, the 100ul/ hole, 37 ℃ of lucifuges developed the color 15 minutes; Use 2mol/L H
2SO
4After the termination reaction, read the OD490 value in each hole.
The result: the Virga monoclonal antibody of hybridoma cell strain secretion is IgG1 subclass immunoglobulin (Ig).
4.3 the purifying of monoclonal antibody
Before the monoclonal antibody purifying, one all needs ascites is carried out pre-treatment, and purpose is in order further to remove cell and residue thereof, finely ground particle substance and fat granule etc.Use filtering with microporous membrane ascites, to remove larger grumeleuse and fat granule; Remove cell residue and finely ground particle substance with 10000g 15 minutes high speed centrifugation (4 ℃).
Ammonium sulfate precipitation method:
Saltout: the ascites that absorption 10ml handles well moves in the small beaker, under agitation, drips saturated ammonium sulphate solution 5.0ml; Continue slowly to stir 30 minutes; Centrifugal 15 minutes of 10000r/min; Abandoning supernatant, throw out suspends with 1/3 saturation ratio ammonium sulfate, and stirring action 30 minutes is centrifugal with method; Repeat back 1-2 time; Throw out is dissolved in 1.5ml PBS (0.01mol/L PH7.2) or the Tris-HCl damping fluid.
Desalination: column chromatography commonly used or dialysis method.Column chromatography is that the sample of saltouing is crossed Sephadex G-50 chromatography column, with PBS or Tris-HCl damping fluid as balance liquid and elutriant, flow velocity per minute 1ml.First protein peak is the antibody-solutions of desalination.
D. the mensuration of protein content:
(Pr) (mg/ml)=(1.45 * OD280-0.74 * OD260) * extension rate; Or (Pr)=OD280 * extension rate/1.3
E. packing is frozen for subsequent use.
4.4 the evaluation of monoclonal antibody
4.4.1 the stability of monoclonal antibody
Recovery hybridoma cell strain 2H5B12C3 (CCTCC NO:C2010123) collects the cell conditioned medium of different passage numbers, and detects it with the ELISA method and tire.The result shows the generation monoclonal antibody that this cell strain can be stable.
4.4.2 the atopic of monoclonal antibody
(1) indirect elisa method (seeing the evaluation of immunoglobulin subclass) detects gained monoclonal antibody and N-acetic acid Virga-BSA antigen-reactive characteristics
Experimental result sees Table 1.The result shows, specific reaction occurs for monoclonal anti physical efficiency and the N-acetic acid Virga-BSA of hybridoma cell strain secretion, and with carrier bovine serum albumin (BSA) no cross reaction.
Table 1.ELISA detects the specific reaction of monoclonal anti antibody and N-acetic acid Virga-BSA
(2) Western Blot immunoblot experiment inspection monoclonal antibody and N-acetic acid Virga-BSA association reaction
N-acetic acid Virga-BSA carries out the 12%SDS-PAGE electrophoresis, in Bio-Rad electrotransfer system, the gel protein band is transferred on the nitrocellulose filter according to a conventional method, spend the night 4 ℃ of sealings with 5% skim-milk, after primary antibodie (Virga monoclonal antibody) is hatched, wash film three times, each 5 minutes, with the sheep anti mouse two with IRDye 800 marks anti-(1: 5000, RocklandImmunochemicals, Boyertown, PA) hatch 2 hours under the room temperature, wash film three times, each 5 minutes, film is placed on Odyssey instrument (LI-COR, Inc., Lincoln, NE) middle scanning and analysis picture.
The result has specific band as shown in figure 10 about 68KD, show that specific reaction occurs for monoclonal anti physical efficiency and N-acetic acid Virga-BSA, and with carrier bovine serum albumin (BSA) no cross reaction.
The result of ELISA and Western Blot proves that the monoclonal antibody of hybridoma cell strain secretion can the specific binding reaction occur with N-acetic acid Virga-BSA, and with carrier bovine serum albumin (BSA) no cross reaction.
The preparation of embodiment 5 colloid gold chromatographic test paper strips
(1) the Virga artificial antigen shown in the preparation formula (I-i) sees embodiment 1 for details;
(2) preparation Virga monoclonal antibody sees embodiment 2 and embodiment 3 for details;
A, preparation contain the mixing solutions of the colloid gold label thing of the colloid gold label thing of anti-Virga monoclonal antibody and mouse IgG monoclonal antibody: with the trisodium citrate reductive agent hydrochloro-auric acid is reduced the colloidal gold solution of making 20nm-40nm; Use 0.1mol/LK
2CO
3Regulate described colloidal gold solution to optimal pH 8.5-9.0, after the Virga monoclonal antibody pure water dialysis of the present invention with purifying, add in the colloidal gold solution by 3 μ g/ml amount, room temperature induction stirring 15-30min, then the amount by 5 μ g/ml adds mouse IgG monoclonal antibody, room temperature induction stirring 15-30min, dropwise add 1% network protein solution, making final concentration is 0.1%, continue to stir 5min, 4 ℃ of centrifugal 10min of lower 15000rpm, supernatant discarded, throw out is returned to the original volume mixing again with resuspended liquid, recentrifuge once is suspended to 1/20,4 ℃ of original volume with resuspended solution with throw out and saves backup, namely obtain to contain the mixing solutions of the colloid gold label thing of the colloid gold label thing of anti-Virga monoclonal antibody and mouse IgG monoclonal antibody, wherein said resuspended liquid is the 0.02Tris-HCL damping fluid that contains 5% marine alga;
(3) the colloid gold label thing that contains anti-Virga monoclonal antibody of dilution in 1: 15 and the mixing solutions spraying of the colloid gold label thing of mouse IgG monoclonal antibody are adsorbed in the trevira felt, 27 ℃ of heat preservation and drynesses, preparation Virga gold-marking binding pad 3, the spraying coating process Simple fast, spraying is even, fast drying.
(4) at the coated orthoscopic detection line T line 4 of Virga artificial antigen solution 0.6mg/ml of coated film 3 usefulness embodiment 1 preparation, be vertically arranged in parallel with coated 5, two lines of orthoscopic control line C line of sheep anti-mouse igg solution 2.0mg/ml.
(5) assembling of test strip (Fig. 1～2): sample pad 1, gold mark combination mat 2, coated film 3 and absorbent pad 6 are sticked on the liner plate 7 successively, sample pad 1 places an end of liner plate 7, absorbent pad 6 places the other end of liner plate 7, coated film 3 places the centre of liner plate 7, gold-marking binding pad 2 places between sample pad 1 and the coated film 3, namely obtain detecting the large plate of test pater for the Virga colloidal gold chromatographic of immunodetection, large plate is cut into the test strip of 2.5mm-7.0mm width specifications by customer requirement, hermetically drying is preserved.
Be respectively Virga solution and the distilled water (being Virga concentration 0mg/ml) of 5ppb, 100 μ g/ml, 500 μ g/ml with the Virga colloidal gold strip detectable level of embodiment 5 preparations, the results are shown in Figure 3, find out that by the result distilled water (containing Virga 0mg/ml) result is negative; The result is positive for 5ppb Virga solution; 100 μ g/ml Virga solution results are positive; 500 μ g/ml Virga solution results are positive; 1mg/ml Virga result is positive.
Be respectively Vardenafil solution and the distilled water (being Vardenafil concentration 0mg/ml) of 5ppb, 100 μ g/ml, 500 μ g/ml with the Virga colloidal gold strip detectable level of embodiment 5 preparations, the results are shown in Figure 4, find out that by the result distilled water (containing Vardenafil 0mg/ml) result is negative; The result is positive for 5ppb Vardenafil solution; 100 μ g/ml Vardenafil solution results are positive; 500 μ g/ml Vardenafil solution results are positive; 1mg/ml Vardenafil result is positive.Illustrate that the Virga colloidal gold test is applicable to have with it detection of the derivative Vardenafil of identical parent nucleus.
Be respectively Acctildenafil solution and the distilled water (being Acctildenafil concentration 0mg/ml) of 5ppb, 100 μ g/ml, 500 μ g/ml with the Virga colloidal gold strip detectable level of embodiment 5 preparations, the results are shown in Figure 5, find out that by the result distilled water (containing Acctildenafil 0mg/ml) result is negative; The result is positive for 5ppb Acctildenafil solution; 100 μ g/ml Acctildenafil solution results are positive; 500 μ g/ml Acctildenafil solution results are positive; 1mg/ml Acctildenafil result is positive.Illustrate that the Virga colloidal gold test is applicable to have with it detection of the derivative Acctildenafil of identical parent nucleus.
Be respectively Tadalafei solution and the distilled water (being Tadalafei concentration 0mg/ml) of 5ppb, 100 μ g/ml, 500 μ g/ml with the Virga colloidal gold strip detectable level of embodiment 5 preparations, the results are shown in Figure 6, find out that by the result distilled water (containing Tadalafei 0mg/ml) result is negative; The result is negative for 5ppb Tadalafei solution; 100 μ g/ml Tadalafei solution results are negative; 500 μ g/ml Tadalafei solution results are negative; 1mg/ml Tadalafei result is negative.Illustrate that the Virga colloidal gold test is not suitable for the detection of Tadalafei.
The structural formula of Virga, Vardenafil, Acctildenafil, Tadalafei is as follows successively:
Virga; Vardenafil
Acctildenafil; Tadalafei
Can find out that by the said structure formula Virga, Vardenafil, Acctildenafil all have identical parent nucleus, and Tadalafei does not have identical parent nucleus with former three, as seen Virga colloidal gold strip of the present invention can be used for detecting Virga and has that non-compounds of identical parent nucleus with Virga, and can not be used for the compound different from the Virga parent nucleus.
The present invention detects the detection reaction principle of the colloid gold chromatographic test paper strip of Virga:
Virga belongs to small-molecule substance, the present invention adopts competition law, i.e. Virga in the sample or have the analog of identical parent nucleus with it and be fixed on the Virga monoclonal antibody of envelope antigen N-acetic acid Virga on the coated film 3-BSA competition colloid gold label.After test strip is with sample pad 1 terminal immersion sample, sample solution passes through from the bottom up swimming of wicking action along test strip, the upper dry anti-Virga gold mark monoclonal antibody of dissolving gold mark pad, if there is not medicine to be measured in the testing sample, then Virga gold mark monoclonal anti is known from experience the N-acetic acid Virga of direct swimming on detection line and the nitrocellulose membrane-BSA immune response is occured, thereby colloid gold particle is assembled, and forms red lines at the detection line place.If there is medicine to be measured in the testing sample, then the Virga gold is marked the detection thing generation immune response in monoclonal anti cognition and the sample and can not be moved forward, owing to not having Virga gold mark monoclonal antibody to be combined with the detection line coating antigen, not occurring thereby detection line does not just have red lines.Control line (5) is coated with sheep anti-mouse igg, no matter whether there is medicine to be measured in the sample, another kind of golden labeling antibody mouse IgG gold mark monoclonal antibody in the gold-marking binding pad all can be combined with sheep anti-mouse igg because capillary action moves to control line (5), immune response occurs, colloid gold particle is assembled, and forms red lines.
Colloid gold chromatographic test paper strip high specificity of the present invention, susceptibility is high, detects minimum limiting the quantity of and can reach 5ppb; Easy, quick, ageing strong, need not any other reagent and instrument, but execute-in-place, test strip can be judged detected result after adding test sample liquid in 5-10 minute; The result shows image, directly perceived, accurate; Cost saving, applied widely, be convenient to promote.
Claims (11)
1. the hybridoma cell strain 2H5B12C3 of strain secretion Virga monoclonal antibody is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:C2010123.
2. a Virga monoclonal antibody is characterized in that by preserving number claimed in claim 1 being the hybridoma cell strain generation of CCTCCNO:C2010123.
3. the application of Virga monoclonal antibody claimed in claim 2 in preparation Virga detection reagent.
4. application according to claim 3 is characterized in that described Virga detection reagent is Virga detection colloid gold chromatographic test paper strip.
5. colloid gold chromatographic test paper strip for detection of Virga, the sample pad (1) that comprises liner plate (7) and on liner plate (7), be connected successively, gold-marking binding pad (2), coated film (3) and absorbent pad (6) form, gold-marking binding pad (2) is the trevira felt of absorption Virga monoclonal antibody colloid gold label thing and mouse IgG monoclonal antibody colloid gold label thing, at the coated orthoscopic detection line T line (4) of the upper useful Virga antigenic solution of coated film (3), with the coated orthoscopic control line C line (5) of usefulness sheep anti-mouse igg solution, article two, line is vertically arranged in parallel, and it is characterized in that described Virga monoclonal antibody colloid gold label thing is the colloid gold label thing of Virga monoclonal antibody claimed in claim 2.
6. the colloid gold chromatographic test paper strip for detection of Virga according to claim 5 is characterized in that described Virga antigen is the Virga artificial antigen.
7. the colloid gold chromatographic test paper strip for detection of Virga according to claim 6 is characterized in that described Virga artificial antigen is:
8. the colloid gold chromatographic test paper strip for detection of Virga according to claim 7 is characterized in that described carrier proteins is bovine serum albumin, chicken ovalbumin or people's hemocyanin.
9. the colloid gold chromatographic test paper strip for detection of Virga according to claim 8 is characterized in that described carrier proteins is bovine serum albumin or chicken ovalbumin.
10. the colloid gold chromatographic test paper strip for detection of Virga according to claim 9 is characterized in that described carrier proteins is bovine serum albumin.
11. the preparation method of the colloid gold chromatographic test paper strip for detection of Virga claimed in claim 7 is characterized in that comprising following steps:
(1) the Virga artificial antigen shown in the preparation formula (1);
(2) preparation Virga monoclonal antibody;
(3) preparation contains the mixing solutions of the colloid gold label thing of the colloid gold label thing of anti-Virga monoclonal antibody and mouse IgG monoclonal antibody;
(4) utilize the mixing solutions of the colloid gold label thing of the described colloid gold label thing that contains anti-Virga monoclonal antibody of step (3) and mouse IgG monoclonal antibody to prepare the gold-marking binding pad of Virga monoclonal antibody;
(5) be coated with orthoscopic detection line T line at coated film with described Virga artificial antigen solution, with the coated orthoscopic control line C line of sheep anti-mouse igg solution, two lines are vertically arranged in parallel;
(6) assembling test strip.
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| CN108752345A (en) * | 2018-03-23 | 2018-11-06 | 广东工业大学 | Silaenafil haptens, artificial antigen and preparation method thereof |
| CN108548925A (en) * | 2018-03-28 | 2018-09-18 | 韶关学院 | Ternary system Immune competition method detects the quantum dot immune chromatography detection card and detection method of silaenafil similar drug |
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